One aspect of this project focuses on resolving the genetics of MH by improving the specificity and sensitivity of MH phenotyping by contracture testing of biopsied muscle. The project will determine if smaller amounts of muscle can be taken from the patient, placed in a skinning solution and shipped to a testing center where single fibers, rather than large bundles, will be tested. The number of single fibers contained in just one of the 6 to 9 fascicles used in current testing is about 200, illustrating how much material is available for single fiber testing. In addition, skinned fibers can be stored for several weeks, and each fiber can be retested several times without significant change to responses. The other aspect of the project will search for a mutation that causes MH in the 50 percent of families in which there is no linkage to chromosome 19. The FKBP12 protein binds avidly to and modulates the RYR1 calcium release channel (CRC). When FKBP12 is dissociated from RYR1, the CRC becomes more sensitive to caffeine-induced channel opening. These effects of FKBP12 on the CRC make it a prime suspect for MH etiology. The applicant will screen for FKBP12 mutations in contracture phenotyped patients and test the functional effects of replacement of native FKBP12 with recombinant, mutant FKBP12 proteins on contracture in single skinned fibers and on calcium release in sarcoplasmic reticulum membrane vesicles.